The ciliate Tetrahymena thermophila excises approximately 6000 specific DNA segments from its somatic nucleus during development. This project aims to understand the regulation of this massive genome reorganization and ultimately learn fundamental principles governing chromosome structure and stability. Understanding how particular DNA segments, called internal eliminated sequences (IES), are selectively excised is challenged by the fact that they share little common structure. Currently, we have limited knowledge of the protein machinery that recognizes and excises these 6000 IES. While four proteins have been linked to this process, their exact roles have yet to be fully elucidated. We have identified five genes encoding novel proteins putatively involved in DNA rearrangement. These were identified using a Green Fluorescence Protein (GFP)-based screening strategy designed to find developmentally expressed proteins that localize specifically to the differentiating nuclei where and when DNA rearrangement occurs. To demonstrate whether these five candidates are required for rearrangement, we will knock out their genes and analyze the phenotype of the resulting transgenic strains. Furthermore, we will test whether these proteins interact with previously identified DNA rearrangement proteins and/or associate with the IES during their excision. Initial characterization of one of these genes, LIA1, indicates that its protein very likely participates in DNA rearrangement. Characterization of additional components of the DNA rearrangement machinery will greatly enhance our understanding of this process. This biological phenomenon provides a unique system with which to discover mechanisms cells use to recognize individual chromosomal segments throughout the genome. Such mechanisms are likely critical for ensuring chromosome stability. Given that cancer cells commonly exhibit aberrant DNA rearrangements, identification of the cellular mechanisms that ensure faithful chromosome maintenance is an important step leading to an understanding of the molecular basis of disease associated with genetic instability.